Pneumococci continue to be the major cause of pneumonia, meningitis, and otitis media in young children and other high risk population worldwide. Pneumococcal polysaccharide (PS), designated as T-cell independent type 2 (TI-2) antigen, induces poor immune response in young children. Splenic marginal zone B cells, associated with CD21, CD19 and C3d, play an important role in TI-2 antibody responses, and provide host defense against bacterial pathogens. We have studied the antibody response, avidity, and opsonophagocytic activity of antisera in mice immunized with type 9V PS conjugated to inactivated pneumolysin (Ply) or autolysin (Aly), and 7-valent pneumococcal conjugate vaccine. Compared to mice given 9V PS alone, serum IgG and IgM concentrations against the 9V PS were higher in mice immunized with conjugates. High concentrations of serum antibodies were maintained for over 12 weeks. The relative avidities of IgG and IgM antibodies and opsonophagocytic activity against 9V pneumococci were high in mice immunized with conjugates. Thus, conjugate vaccines can induce high as well as long duration of antibody response and effective functional activity. In another study, mice received intranasal immunization with type 9V conjugate or 9V PS. These animals produced high concentrations of 9V PS IgG and IgA antibodies in their serum, spleen, intestine, lung, Peyer's patch and fecal extract samples. Mice immunized with these glycoconjugates exhibited opsonophagocytic activity and rapid bacterial clearance from blood and provided homologous and cross-protection against challenge with virulent pneumococci. These results indicate that intranasal immunization with glycoconjugate vaccines may serve as an alternative and convenient approach for prevention of pneumococcal infection. Most antigens are poor mucosal immunogens and need effective adjuvants. Mucosal immune responses against pneumococcal conjugates have been conducted using CpG oligodinucleotide (ODN). The adjuvant activity of CpG ODN may associate with the binding of CpG to the Toll-like receptor 9, induction of Th1-inducing cytokines, MHC and costimulatory molecules of antigen presenting cells. High IgG and IgA antibody responses were observed in the samples including serum, Peyer's patch, and intestine of mice immunized intranasally with 9V PS-Ply conjugate. In contrast, no significant difference in antibody responses were observed between mice given 9V PS and non-immunized control group. These results suggest that CpG could serve as an effective adjuvant to induce mucosal immunity for pneumococcal conjugate vaccine. Different bacterial vaccines including Haemophilus b conjugate, pneumococcal type 6 B PS, and typhoid Vi PS vaccines were irradiated with various doses of gamma, electron beam and X-ray. In general, there is correlation between the irradiation dosage and increased values of the Kd, and therefore, decreased the molecular size and stability of these vaccines. Quality control of pneumococcal Polysaccharide-protein conjugate vaccines. Protection against pneumococcal infection depends on the presence of antibodies against capsular polysaccharides (PSs). These serotype-specific antibodies facilitate phagocytosis to destroy invading bacteria. The spleen is an important organ in the immune response to pneumococci, since it contains both antibody-producing B-cells and phagocytes. The 23-valent pneumococcal PS vaccine is effective against bacteremic pneumococcal disease in adults and is cost-effective for routine immunization of the elderly. However, the PS vaccine is not sufficiently immunogenic in infants and young children. Chemical coupling of a PS to a carrier protein to form a glycoconjugate greatly improves the immunogenicity of a PS. The 7-valent pneumococcal conjugate vaccine, Prevnar, appears to be highly effective in preventing invasive disease in young children and to have a significant impact on otitis media. Studies were conducted in our laboratory to evaluate the avidity, antibody response and opsonophagocytic activity of serum antibodies to the pneumococcal type 9V PS conjugated to inactivated pneumolysin (Ply) or autolysin (Aly) and in the 7-valent PS-CRM197 conjugate vaccine. The effect of priming mice with the 7-valent conjugate followed by a booster dose of 23-valent PS vaccine was also examined. Compared to 9V PS alone or non-immunized controls, the serum concentrations of 9V PS IgG and IgM antibodies were significantly higher in mice immunized with 9V PS-Ply, 9V PS-Aly conjugates, or 7-valent conjugate vaccine at 0, 2, and 4 weeks, 7-valent conjugate followed by 23-valent PS vaccine compared to 9V PS alone. Thus, priming mice with polyvalent conjugate vaccine induced high 9V PS antibody response in contrast to the group primed with polyvalent PS vaccine. The duration of antibody response in mice was studied in mice immunized with various conjugate vaccines and boosted with 23-valent PS vaccine. The antibody response was highest at 4 weeks after the injection, increasing 7-10 fold compared to the group that received 9V PS alone. The high IgG Ab response was maintained for over 12 weeks after final injection. Similar results were also observed in 9V PS IgM Ab response. In addition, high IgG and IgM Abs were maintained for over 8 weeks after final injection in mice given a booster dose of 23-valent PS vaccine. The avidity indeces of IgG antibody were highest in mice immunized twice with the 7-valent conjugate vaccine, and combined immunization with 7-valent conjugate then 23-valent PS vaccine, followed by monovalent 9V PS-protein conjugates. Mice immunized with 9V PS alone exhibited the lowest avidity. Similar results were observed in the avidity of IgM antibody. The 9V PS-Ply and 9V PS-Aly immunized group showed the opsonophagocytic titers of 16 compared to 4 in 9V PS-immunized and complement control groups. Mice immunized twice with 7-valent conjugate or conjugate vaccine followed by 23-valent PS vaccine showed titers of 32 and >64 respectively. These results indicate that monovalent and 7-valent conjugate vaccines can stimulate effective functional activity that correlated with protective immunity against pneumococcal infection. Pathogenic pneumococci enter the body through the respiratory mucosa and may cause serious infections such as pneumonia, bacteremia, and meningitis in high risk population. They are also a common cause of mucosal infection, e.g. otitis media and sinusitis in young children. The potential of mucosal immunity to protect against pneumococcal infection was studied in mice through intranasal immunization using pneumococcal type 9V PS and 9V glycoconjugates. Compared to the non-immunized control group, concentrations of 9V IgG and IgA antibodies in serum, spleen, intestine, lung, Peyer's patch and fecal extract samples were significantly higher in mice immunized with 9V PS-Ply, or 9V PS-Aly, as well as 9V PS alone. The opsonophagocytic titers in serum and intestine homogenate samples were significantly higher in the immunized groups compared to the complement control group. Furthermore, the mice immunized with 9V PS-Ply, 9V PS, or 7-valent conjugate vaccine showed more rapid bacterial clearance from blood at 1, 3, and 5 hrs after challenge with 9V pneumococci compared to the non-immunized control group. Mice immunized with 9V PS-Ply, or 7-valent conjugate vaccine also showed more rapid bacterial clearance after challenge with 19F pneumococci compared to the non-immunized control group. These functional data thus indicate that mice immunized with PS-protein conjugate vaccines had high serum opsonophagocytic activity, rapid bacterial clearance from blood, and provided cross- protection against tested heterologous pneumococcal serotype. Most antigens are poor mucosal immunogens and need effective adjuvants. A number of mucosal adjuvants have been proposed, including bacterial toxin, such as cholera toxin and E. coli heat-labile enterotoxin, and CpG oligodeoxydinucleotide (ODN), In present study, CpG ODN was used as adjuvant. High IgG and IgA antibody responses were observed in the samples including serum, Peyer's patch, and intestine of mice immunized intranasally with 9V PS-Ply conjugate. In contrast, no significant difference in antibody responses were observed between mice given 9V PS and non-immunized control group. These results suggests that CpG could serve as an effective adjuvant to induce mucosal immunity for pneumococcal conjugate vaccine. Gamma radiation has been used for the terminal sterilization and viral inactivation of the biological products. In present study, different bacterial vaccines including Haemophilus b conjugate, pneumococcal type 6B PS, and typhoid Vi PS vaccines were irradiated with various doses of gamma, electron beam and X-ray. In general, there is correlation between the irradiation dosage and increased values of the Kd, and therefore, decreased the molecular size and stability of these vaccines.